The present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology. The invention relates to the fields of agriculture, animal husbandry, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary technology.
Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Erythromycin, FK-506, FK-520, megalomycin, narbomycin, oleandomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; 97/02358; and 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; and 5,712,146; Fu et al., 1994, Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34(8): 881-888, each of which is incorporated herein by reference.
Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes. These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. PKS enzymes are encoded by PKS genes that usually consist of three or more open reading frames (ORFs). Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis. These two major types of PKS enzymes are commonly referred to as Type I or xe2x80x9cmodularxe2x80x9d and Type II xe2x80x9citerativexe2x80x9d PKS enzymes. A third type of PKS found primarily in fungal cells has features of both the Type I and Type II enzymes and is referred to as xe2x80x9cfungalxe2x80x9d PKS enzymes.
Modular PKSs are responsible for producing a large number of 12-, 14-, and 16-membered macrolide antibiotics including erythromycin, megalomycin, methymycin, narbomycin, oleandomycin, picromycin, and tylosin. Each ORF of a modular PKS can comprise one, two, or more xe2x80x9cmodulesxe2x80x9d of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three (for the simplest extender module) or more enzymatic activities or xe2x80x9cdomains.xe2x80x9d These large multifunctional enzymes ( greater than 300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying xcex2-carbon processing activities (see O""Hagan, D. The polyketide metabolites; E. Horwood: New York, 1991, incorporated herein by reference).
During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmid-based Streptomyces coelicolor expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al., 1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262: 1546-1557, and U.S. Pat. Nos. 5,672,491 and 5,712,146, each of which is incorporated herein by reference). The advantages to this plasmid-based genetic system for DEBS are that it overcomes the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythraea, allows more facile construction of recombinant PKSs, and reduces the complexity of PKS analysis by providing a xe2x80x9ccleanxe2x80x9d host background. This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication No. WO 98/49315, incorporated herein by reference).
The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of xcex2-carbon processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, 1998, Curr. Opin. Microbiol. 1: 319-329; Carreras and Santi, 1998, Curr. Opin. Biotech. 9: 403-411; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference). This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters.
One example of this technology involves the use of a PKS in which the first extender module is inactivated by mutation and synthetic molecules, called diketides. These diketides are provided to the altered PKS and bind to the second extender module. The diketides are then processed by the PKS in the normal fashion to yield a polyketide. If the diketide provided differs in structure from the corresponding diketide that is the product of the first extender module, then the polyketide will correspondingly differ from the natural polyketide produced by the intact PKS. See PCT patent publication Nos. 97/02358 and 99/03986, each of which is incorporated herein by reference. One important compound produced by this technology resulted from feeding a propyl diketide to DEBS to produce 15-methyl-6-dEB. This molecule is referred to herein as propyl-6-dEB, because it has a C-13 propyl group where 6-dEB has a C-13 ethyl group.
While the diketide feeding technology provides useful amounts of compound, the cost of producing polyketides by that technology is increased by the need to prepare the synthetic diketide. Moreover, certain polyketide producing cells degrade some of the diketide before it can be incorporated into a polyketide by the PKS, thus increasing the cost of production. Thus, there remains a need for methods to produce polyketides by other means. The present invention helps meet that need by providing recombinant host cells, expression vectors, and methods for making polyketides in diverse host cells.
The present invention provides recombinant host cells and expression vectors for making propyl-6-dEB and compounds derived therefrom. The present invention also provides methods for increasing the amounts of propyl-6-dEB produced in a host cell by providing recombinant biosynthetic pathways for production of a precursor utilized in the biosynthesis of the compound and optionally altering other biosynthetic pathways in the cell.
In one embodiment, the host cell does not produce propyl-6-dEB, and the host cell is modified by introduction of a recombinant expression vector so that it can produce the compound. In another embodiment, propyl-6-dEB is produced in the host cell in small amounts, and the host cell is modified by introduction of a recombinant expression vector so that it can produce the compound in larger amounts. In a preferred embodiment, the host cell is altered to produce the precursor butyryl CoA by transferring the genes that encode the enzymes that produce butyryl CoA from a first cell to the host cell. The transfer is accomplished using an expression vector of the invention. The expression vector drives expression of the genes and production of butyryl CoA in the second cell.
In another embodiment, the product is a polyketide other than 6-dEB that is made by a PKS that utilizes propionyl CoA. The polyketide produced by the host cell of the invention containing the PKS differs from the usual product produced by the PKS in that butyryl CoA instead of propionyl CoA is utilized by the PKS in producing the polyketide. The polyketide is a polyketide synthesized by either a modular, iterative, or fungal PKS. In one preferred embodiment, the polyketide is synthesized by a modular PKS.
In one embodiment, the host cell is either a procaryotic or eukaryotic host cell. In one embodiment, the host cell is a Saccharopolyspora host cell, including but not limited to S. erythraea. In another embodiment, the host cell is a Streptomyces host cell, including but not limited to S. coelicolor, S. lividans, and S. venezuelae. In another embodiment, the host cell is an E. coli host cell. In another embodiment, the host cell is a yeast host cell. In another embodiment, the host cell is a plant host cell.
In one embodiment, the invention provides a recombinant expression vector that comprises a promoter positioned to drive expression of one or more genes that encode the enzymes required for biosynthesis of butyryl CoA. In a preferred embodiment, the promoter is derived from a PKS gene. In a related embodiment, the invention provides recombinant host cells comprising one or more expression vectors that drive expression of the enzymes that produce the precursor.
In another embodiment, the invention provides a recombinant host cell that comprises not only an expression vector of the invention but also an expression vector that comprises a promoter positioned to drive expression of a PKS. In a related embodiment, the invention provides recombinant host cells comprising the vector that produces the PKS and its corresponding polyketide.
These and other embodiments of the invention are described in more detail in the following description, the examples, and claims set forth below.